A selective CuII complex with 4-fluorophenoxyacetic acid hydrazide and phenanthroline displays DNA-cleaving and pro-apoptotic properties in cancer cells

The thin line between efficacy and toxicity has challenged cancer therapy. As copper is an essential micronutrient and is important to tumor biology, CuII complexes emerged as an alternative to chemotherapy; however, its biological properties need to be better understood. Thus, we report in vitro the antitumor effects of two CuII complexes named [Cu(4-fh)(phen)(ClO4)2] (complex 1) and [Cu(4-nh)(phen)(ClO4)2]·H2O (complex 2), in which 4-fh = 4-fluorophenoxyacetic acid hydrazide; 4-nh = 4-nitrobenzoic hydrazide and phen = 1,10-phenanthroline. Both complexes presented cytotoxic activity against tumor cells, but only complex 1 showed significant selectivity. Complex 1 also induced DNA-damage, led to G0/G1 arrest and triggered apoptosis, which was initiated by an autophagy dysfunction. The significant in vitro selectivity and the action mechanism of complex 1 are noteworthy and reveal this prodrug as promising for anticancer therapy.

The understanding of pharmacological properties of different substances, associated with the evolution of laboratory techniques related to the bioinorganic profile, allowed the industrial-scale synthesis of molecules with antiinflammatory, anticancer and antibacterial properties 1 . In this context, previous studies have shown that metal complexes bearing hydrazides and derivatives are generally more effective than their respective free ligands [2][3][4][5][6][7] . For example, a series of Pt II -hydrazide complexes present higher inhibitory effect towards Friend leukemia cells in comparison with their respective free ligands and cisplatin 8,9 . Pt II complexes bearing cyclopentanecarboxylic acid hydrazide induce apoptosis and exert significantly lower in vivo toxicity as compared with cisplatin 10 . In fact, metal complexes bearing hydrazides have a great pharmacological potential to be explored and the chelation of these ligands with metal ions plays an important role in the improvement of biological properties 7 .
Copper is an essential trace element for the function of enzymes involved in DNA synthesis, energy metabolism and respiration in all living organisms 11 . These properties have been taken into account for the design of new copper complexes with antitumor action, since endogenous metals may be less toxic 1,12 . Special attention has been devoted to Casiopeinas, which have entered phase I clinical trials in Mexico 13,14 . Among them, Cas III-ia induces cell death by autophagy and apoptosis, through the activation of reactive oxygen species (ROS)

Results
Cytotoxicity studies. Two complexes ( Fig. 1) were prepared according to the procedure previously published by our group 16 . The X-ray structural analysis and EPR data confirmed that the geometry around the Cu II ion is octahedral distorted, with two perchlorate anions occupying the apical positions. Nevertheless, conductivity measurements revealed that complexes of the type [Cu(N-O)(N-N)] 2+ are produced in a solution. We also verified the stability of these species, an important factor for the development of clinical metal-based drugs, and they were stable in a mixture containing H 2 O/DMSO 0.1% with or without buffer for at least 6h 16 . In previous in vitro assays performed by our group, it was found that these complexes (1 and 2) displayed high cytotoxic activity, being more active than carboplatin, free ligands and [Cu(phen) 2 ] 2+ towards K562, MDA-MB-231 and MCF-7 cell lines. These studies also indicated that 1 and 2 induce apoptotic cell death in MDA-MB-231 cell line and bind to DNA with K values of 4.38 × 10 4 and 2.62 × 10 4 , respectively 16 . Considering these aspects, the evaluation of these compounds regarding selectivity is highly desirable, as well as the understanding of their mechanisms of action in detail.
As demonstrated in Table 1, the resazurin reduction assay demonstrated that both complexes were active against the Sarcoma 180 cell line displaying IC 50 values comparable to cisplatin. It is interesting to note that complex 1 was slightly more cytotoxic than complex 2, with IC 50 value of 4.16 µM. Although complex 2 also presented high cytotoxicity against Sarcoma 180 cell line (IC 50 Sarcoma 180 = 5.15 µM), it was not selective (SI < 2). Thus, complex 1 was selected for further studies because it showed excellent selectivity index values against Sarcoma 180 cell line (IC 50 = 4.16 µM and SI = 4.77) and expressed high activity and selectivity against the BF16F10 melanoma cell line (IC 50 = 1.83 µM and SI > 10). The IC 50 observed in the non-tumoral myocyte C2C12 cell line was much higher than the one observed in tumoral cell lines (IC 50 = 19.85).
Cell death induced by copper complexes has been previously associated with increased levels of ROS. Hence, we used quercetin, a known ROS scavenger, to verify the effects of complex 1 in the oxidative pathway. Sarcoma 180 and melanoma B16F10 were first treated with quercetin for 24 hours, which was cytotoxic only for melanoma B16F10 (IC50 = 110.4 µM) (see Supplementary Fig. S1 online). Subsequently, both cells were treated with complex 1 alone and associated with quercetin for 24 h. The IC 50 for sarcoma 180 cells increased to 19.82 µM (Fig. 2), when these cells were treated with quercetin, indicating the ability of quercetin to attenuate cytotoxicity induced by complex 1, probably due to ROS inactivation. This behavior was not observed for melanoma B16F10 cell line (Fig. 3), whose IC 50 did not increase significantly when these cells were treated with quercetin.
Oxidative stress studies. ROS generation. Sarcoma 180 and melanoma B16F10 cell lines were treated with complex 1 at different concentrations, being 2.5 µM and 10 µM for sarcoma 180, and 1.5 µM and 5.5 µM for melanoma B16F10. Treatments were performed with complex 1 alone or associated with quercetin at 62.5 µM (Fig. 4). In sarcoma 180, complex 1 at 2.5 µM significantly increased ROS levels and quercetin reversed this ef-  Figure 2. Sigmoidal dose-response curves of sarcoma 180 treatment with complex 1 alone (black points) and complex 1 associated with quercetin (red points) for 24 h in the resazurin reduction assay. Each point represents the means ± standard deviation for n = 4 replicates.

Figure 3.
Sigmoidal dose-response curves of melanoma B16F10 treatment with complex 1 alone (black points) and complex 1 associated with quercetin (red points) for 24 h in the resazurin reduction assay. Each point represents the means ± standard deviation for n = 4 replicates. www.nature.com/scientificreports/ fect (Fig. 4A). In melanoma B16F10, complex 1 at 5.5 µM significantly increased ROS levels, but the antioxidant quercetin did not reverse this effect (Fig. 4B). Actually, quercetin induced the ROS production in melanoma B16F10 when treated with 5.5 µM of complex 1 and 62.5 µM of quercetin. In both cells, cisplatin induced the ROS production, but quercetin only reversed this effect in sarcoma 180 cells.
Lipid peroxidation. In order to measure the extent of lipid peroxidation induced by complex 1, the levels of Thiobarbituric Acid Reactive Substances (TBARS) were measured in sarcoma 180 cells treated with concentrations of 1 µM and 4 µM and melanoma B16F10 cells treated with 1.5 µM and 5.5 µM. As shown in Fig. 5, com- Data are presented as means ± the standard deviation. *p < 0.05. **p < 0.01, ***p < 0.001 and ****p < 0.0001. The comparison with the negative control was performed by one-way ANOVA followed by Dunnett's post-test and the concentrations with or without quercetin were compared to each other by one-way ANOVA followed by Tukey´s post-test. Plasmid DNA cleavage assay. In order to evaluate the plasmid cleavage capacity of complex 1, siSTRIKE plasmid (30 ng) was treated with different concentrations of the compound, in the presence or absence of H 2 O 2 and DMSO. Complex 1 interacted with plasmid DNA causing breaks (Fig. 6). Gel electrophoresis detected the pres-  www.nature.com/scientificreports/ ence of three band patterns corresponding to the plasmid DNA conformations (Fig. 6B). Form I (supercoiled) refers to the most compact form of the plasmid, without single breaks in the DNA strands, which makes it easier to migrate through the gel network. Form II is the structure regarding breaks in one DNA strand, which makes the conformation more circular or relaxed, being identified as the most superior band of the gel. Form III would be the result of double-strand breaks at DNA molecule, which turns it into the linearized conformation, acquiring an intermediary position in the gel. When plasmid was treated only with complex 1 the intensity of the band corresponding to plasmid form II (circular) increased at all concentrations of the compound when compared to the negative control (no treatment) (Fig. 6B). When complex 1 was associated with DMSO, which is a free radical scavenger, the band corresponding to form I (supercoiled-intact plasmid) increased, demonstrating that DMSO attenuates the cleavage capacity of the complex.
The restriction enzyme NheI was used as a positive control for DNA double-strand breaks, since the plasmid contains a restriction site for this enzyme. An intermediary band was generated after restriction, corresponding to form III (linear) (Fig. 6B). It was also possible to notice the appearance of small bands referring to form III in treatments that involved the isolated complex 1 (5 and 20 µM) and in the lowest concentration of the complex 1 (5 µM) associated with DMSO, which indicates that the complex 1 is also capable of inducing double breaks on the DNA strands. The treatment with only H 2 O 2 (15 mM) simulated an oxidative environment and was responsible for the increase in plasmid form II (circular) (Fig. 6B). However, by associating complex 1 with H 2 O 2 , a total plasmid DNA degradation was observed at the concentrations 5 µM and 20 µM of complex 1 (Fig. 6B). Finally, when this set was associated with DMSO, the lowest concentration of the complex (5 µM) had its action attenuated, since plasmid forms II and III were observed. Thus, the use of DMSO protects DNA from complex 1 damage.
In addition, it is noticeable that the complex 1 causes greater damage to the DNA when in the presence of reactive oxygen species, since the total degradation of plasmid DNA occurred in treatments that included hydrogen peroxide associated with the complex 1 and this situation was reversed when the lowest concentration of the complex was associated with DMSO, reappearing plasmid forms III and II.
Cell cycle arrest and apoptosis activation analysis. In order to assess the effect of complex 1 in the cell cycle, Sarcoma 180 cells were treated with three different concentrations of complex (1 µM, 4 µM and 8 µM) and cell cycle phases were analyzed with flow cytometry. Sarcoma 180 cell cycle was trapped in G0/G1 phase, demonstrating an antiproliferative potential of the complex 1 ( Fig. 7A-E). Cell cycle arrest is a response to DNA damage events and may trigger apoptosis. In this context, we treated melanoma B16F10 and sarcoma 180 with complex 1 and performed qPCR to quantify mRNA relative levels of two genes involved in the apoptosis pathway, B-cell lymphoma 2 (BCL-2) and caspase 3 (CSP3), and two genes of the autophagic pathway (ATG5 and ATG7), once autophagy can also trigger apoptosis.
After treatment with complex 1, BCL-2 transcript levels were downregulated in melanoma B16F10 (Fig. 7F) in both tested concentrations. CSP3 expression was upregulated (Fig. 7G) in the higher concentration, indicating that the apoptotic cascade was activated. In Sarcoma 180, BCL-2 mRNA levels were upregulated at the lower concentration of complex 1 (Fig. 7H) and higher levels of CSP3 were identified when cells were treated with a higher concentration of complex 1 (Fig. 7I).
In light of the fact that the treatment with complex 1 generated ROS, induced genomic and plasmid DNA degradation and arrested cells in G0/G1, we investigated whether complex 1 could be effective in a human tumor cell. In this way, we evaluated by which mechanism cell death of HeLa tumor cells was induced when treated with complex 1. Since the overexpression of the autophagic genes ATG5 and ATG7 in sarcoma 180 cells was induced, we performed monodansylcadaverine staining to verify whether the formation of autophagic vacuoles was induced by the treatment with complex 1 at 1 μM, 4 μM and 20 μM. The Complex 1 at the concentration of 4 µM truly activated autophagic pathway in sarcoma 180 tumor cells, once autophagic vacuoles were detected through monodansylcadaverine staining (Fig. 8A,B).

Discussion
Both complexes synthesized showed cytotoxic properties, but in preliminary tests, complex 1 demonstrated better results such as a high selectivity index, in addition to interesting antitumor properties, making it a candidate for antitumor drugs. The ligands phen and 4-fh or 4-nh did not present significant activity against diverse tumorigenic and non-tumorigenic cells 16,22 . Thus, the chelation to copper ion provided the cytotoxic activity of these compounds.
The Complex 1 demonstrated high rates of selectivity for the Sarcoma 180 and B16-10 tumor cell lines (respectively 4.78 and 10.85), once selective indices are considered as significant when ≥ 3 23,24 , which makes it particularly interesting considering the need for less toxic drugs for tumor treatment. Therefore, complex 1 was selected for further studies. Additionally, when compared to other Cu II complexes containing analogous ligands,   www.nature.com/scientificreports/ complex 1 has already been shown to stand out as one of the most active copper-based compounds 25,26 with high selectivity 22 , as it can be confirmed by the present work. Copper complexes can also generate ROS, inducing DNA damage, inhibiting proteasome function 27 , and triggering apoptosis or other cell death pathways 22,[28][29][30][31][32] . For these reasons, those mechanisms were further analyzed in order us to describe the biological activities of complex 1. Regarding the potential of complex 1 in generating ROS, we performed cytotoxicity tests using quercetin, a known antioxidant molecule which has already been described as playing a protective role against copper(II)-induced oxidative stress 33 . We observed that the co-treatment of complex 1 with quercetin increased the IC 50 of sarcoma 180 cells, demonstration that the ROS generation plays an important role in the complex 1 effect in these tumor cells. However, quercetin did not protect melanoma B16F10 cells from the cytotoxicity effect of our compound; quite the opposite, quercetin significantly enhanced the cytotoxicity of complex 1 in melanoma B16F10. This phenomenon can be explained by the already described melanocytotoxicity exhibited by quercetin, which enhances melanogenesis in melanocytes by increasing the activity and synthesis of tyrosinase. We confirmed this behavior in a cytotoxicity assay with quercetin in both cell lines, in which only melanoma B16F10 cells presented cytotoxicity in the tested concentrations (ranging from 2000 to 3.9 µM) showing an IC 50 of 110.4 µM [34][35][36] .
The Complex 1 exhibited the ability to induce the elevation of intracellular ROS in Sarcoma 180 and melanoma B16F10 cell lines. ROS plays important roles within the cell regulation, cell cycle and cell death 22,[37][38][39] . When intracellular levels of ROS are imbalanced, serious damages to biomolecules can be caused 40 , such as proteins, lipids, nucleic acids and whole organelles 41,42 . Many chemotherapeutic drugs increase intracellular generation of ROS, which brings antiproliferative effect 43 and induces apoptosis 37 . Previous studies have shown that Casiopeinas, which are already undergoing clinical tests, also act through oxidative mechanisms causing related cell damage and death 44 . These biological activities were also observed for the complex 1. Copper complexes are widely described as ROS inductor agents 22,29,31,43,45 , thus activating autophagic pathway, culminating into apoptosis 22,31,45 .
Lipid peroxidation is a key indicator of oxidative stress. Our results indicate that the increased ROS production induced by complex 1 results in oxidative damage and disruptions in redox homeostasis in the cell. The involvement of lipid peroxidation in the cytotoxicity of copper complexes has been reported 46 . Excessive oxidation of lipids alters the physical properties of cellular membranes and other intracellular structures. Moreover, lipid peroxides are also able to propagate further generation of ROS, or degrade into reactive compounds that can cause covalent modification of proteins and DNA 47 , leading to cell death through apoptosis and autophagy mechanisms 48 .
The DNA is responsible for regulating several cellular functions and if this molecule gets damaged, cell death pathways are triggered 27 . In the literature, DNA fragmentation has been reported for many copper(II) complexes containing 1,10-phenanthroline 22,39,40 . These studies are based on the discovery that [Cu(phen) 2 ] + is able to bind/intercalate, and promote DNA cleavage 16,49 . This ability observed for several copper complexes bearing 1,10-phenanthroline may explain the results for complex 1 in the fragmentation of the genomic DNA of Sarcoma 180 tumor cells and in the plasmid DNA cleavage activity.
The data herein obtained demonstrate that complex 1 is effective in damaging plasmid DNA, but with greater activity in an environment rich in free radicals. Thus, the complex is likely to interact with the DNA and cause breaks through ROS induction, via redox mechanisms, corroborating with several other authors 22,50-57 .
Moreover, copper complex associated with 1,10-phenanthroline has already been described in the literature, exhibiting dose-dependent response, and its possible mechanism of action is influenced by free radicals, such as ROS, suggesting the oxidative pathway of copper redox cycling as responsible for DNA damage 22,55 . Souza et al. proposed that the ability to cleave DNA through the oxidative pathway is controlled by the type of N,N-donor linked to the complex 22,52 .
According to Simunkova et al., in oxidative environments such as in the presence of H 2 O 2 (a condition that stimulates the Fenton reaction), dose dependence was observed in the nuclease activity of the copper (II) complexes and total conversion of the supercoiled plasmid (form I) into linear (form III) and circular (form II) plasmids. Furthermore, as observed in our results, all copper complexes studied by these authors were also able to cleave plasmid DNA, even in the absence of H 2 O 2 . The authors suggested that in addition to redox cycling, copper complexes also induce DNA breaks by other mechanisms, such as intercalation 55 .
We also believe that complex 1 has redox cycling as one of the mechanisms of action responsible for DNA damage and also that the Fenton reaction is also responsible for ROS generation; in this case, the hydroxyl radical attacks the DNA inducing single or double breaks, as shown in the equation below: Heinrich et al. 53 used ascorbate as a reducing agent to inhibit the DNA cleavage capacity of copper(II) associated with phenanthroline, proving that its action is mediated by an oxidative mechanism. Several copper complexes analyzed by Singh et al. 56 also exhibited chemotherapeutic effects via oxidative, hydrolytic, induction of conformational changes in the DNA molecule and also by the induction of excessive ROS production. According to the authors, these variations in the action mechanisms are due to the flexible redox behavior of copper 53,56 .
Şenel et al. also used hydrogen peroxide as an activator of the copper(II) complexes and observed the same induction of plasmid cleavage, whereas the addition of DMSO as a hydroxyl radical scavenger induced a reduction in the nuclease activity of copper(II) complexes, suggesting the involvement of the hydroxyl radical in the DNA damage pathway. They also emphasize that the presence of aromatic rings, such as those from 1,10-phenanthroline, adequately serve as a partial responsible for the interaction between the complex and the plasmid 54 .
Copper complexes with Schiff bases have shown oxidative damage to DNA and a mechanism based on Fenton-type reaction has also been suggested (ZEHRA; ROISNEL; ARJMAND, 2019). According to Daravath Our oxidative stress experiments showed that complex 1 induced ROS generation and lipid peroxidation. The oxidative intracellular environment is very harmful and can be directly associated to the DNA damage. In fact, copper is a redox active transition metal and copper complexes are widely known to affect redox cycling 58 .
Chemotherapeutic agents are known for their ability to alter the proliferation of tumor cells and Complex 1 was able to trap Sarcoma 180 cells in G0/G1 phase, demonstrating its antiproliferative potential (Fig. 6). Cells can be in a stationary state (G0) or in a division cycle, which can be divided into four phases (G1, S, G2 and M). Cell cycle is controlled by checkpoints, which ensure cell integrity. If any environmental or molecular problem is detected, the advancement of the cell cycle is blocked 59 . Generally, cells that suffer double-or single-strand breaks in DNA are paralyzed in the G0/G1 or G2 phases 60 . Several copper complexes exhibit the ability to degrade tumor cell DNA 22,61,62 , and such ability was detected in complex 1. The damage on DNA is believed to be responsible for this cell cycle arrest. Many other copper complexes are also described as having such antiproliferative potential 22,[29][30][31] trapping cells in G0/G1 and G2.
In addition to the cytostatic capacity, another desirable characteristic for new chemotherapy drugs is the induction of death in a programmed and controlled way, such as apoptosis 32 . In order to evaluate the potential of the apoptosis induction of complex 1, melanoma B16F10 and sarcoma 180 were treated with the complex and the genes from the apoptotic pathway BCL-2 and Caspase 3 had their expression analyzed with real-time PCR. The downregulation of BCL-2 and the upregulation of Caspase 3 observed in melanoma B16F10 cells indicate that these cells underwent apoptosis after complex 1 treatment. Caspases are initiating and executing proteins of apoptosis. They are present in the cytosol as inactive pro-enzymes, becoming active after proteolytic cleavage. There are two types of caspases, initiators, and effectors, and caspase 3 is considered effector, that is, it results in the apoptotic process 63,64 .
In sarcoma 180, the gene of caspase 3 was also upregulated at the 1.5 µM treatment, but differently from the observed in melanoma B16F10, the expression of BCL-2 was upregulated. An explanation for this event is that in mice, overexpression of the protooncogene BCL-2 attenuates the generation of reactive oxygen species 65 . In the literature, it is known that ROS concentration is intrinsically higher in sarcoma 180 cell line 66 and we have compared ROS levels between these two cell lines, detecting levels of ROS four times higher in sarcoma 180 than in melanoma B16F10 (See supplementary Fig. S4 online). This high concentration of ROS observed in sarcoma 180 may be related to a more dynamic facility to express BCL-2 to relieve oxidative stress in cases of free radical elevation.
In order to find out whether complex 1 would be effective against a human tumor lineage, we used HeLa cells. Complex 1 proved to be a potent apoptosis inducer in tumor cells, as it was observed in many other copper complexes that exhibit this property 22,29,32,61,62 , which is the safest mechanism of action of a drug, since apoptosis is a controlled event 30 . The genomic DNA degradation event triggered by complex 1 is believed to be the responsible for the induction of apoptosis, as described by other authors 22,31,45 .
One of the strategies to overcome the limitation of already-existing chemotherapy treatments is to explore the activation of ROS-dependent autophagy, which may end up in directing the apoptotic pathway 45 . The complex 1 caused relevant oxidative stress in Sarcoma 180 tumor cells, what motivated us to investigate whether 1 would be able to activate the autophagic pathway in these cells. Morphologically, this type of cell death is marked by massive cytoplasmic vacuolization 67,68 since, during the process, intracellular and extracellular substrates are delivered to the lysosomes for degradation or recycling 69,70 . Studies demonstrated that autophagy is a mechanism to alleviate cellular stress. However, when overcoming a certain point there is hyperactivation of this system, and apoptotic pathway is activated 45,71,72 . Complex 1 behaved similarly to other copper complexes that induce hyperactivation of the autophagic pathway 22,31,45 . It has been described that almost all classes of anticancer therapy, including agents that cause DNA damage, affect the autophagy mechanism 73 .
Taken together, we summarized in Fig. 9 a proposed mechanism of action of complex 1. In summary, we here demonstrated the in vitro antitumor activity of complex 1 highlighting its cytotoxic effect on cancer cell lines. The chemical compound elicited cell cycle arrest, promoted ROS generation, induced genomic DNA damage and triggered cell death through apoptotic and autophagic mechanisms. Complex 1 showed high selectivity in vitro, making it a promising candidate as chemotherapeutic agent. Copper as chemotherapeutic agents has been explored and we suggest that the combination of redox-active molecules, such as complex 1, with conventional therapy can be useful to improve oncologic treatments and overcome resistance mechanisms. However, additional assays are needed to verify the anti-tumor efficacy of complex 1 in other animal models, to assess its toxicity and mechanism of action.

Methods
Starting materials. All chemicals were purchased from Merck and were used as received unless otherwise stated. Doxorubicin hydrochloride (DXR) was used in the present study as a positive control at 0.4 mM (diluted in autoclaved reverse osmosis water) for in vivo tests. DXR at 0.4 mM was previously able to generate ROS and induce homologous recombination in Drosophila melanogaster through topoisomerase inhibition 74-77 . Preparation of the Cu(II) complexes. The complexes 1 and 2 already described in the literature were prepared according to the published procedure 16 . Elemental analysis (CHN) was used to verify the purity of these complexes.   Reverse transcription and qPCR. After treatments, the total RNA of each sample was extracted using the TRIzol Reagent (Life Technologies), following the manufacturer's recommendations. Quantification was performed in a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and the 260 nm/280 nm ratio was also used as a qualitative parameter. Then, total RNA was stored at − 80 °C for subsequent analyses. For reverse transcription, 2 μg of total RNA was denatured at 70 °C for 5 min, cooled on ice for 5 min and added to a mix containing 10 μL of GoScript Reverse Transcription Oligo(dT) (A2791, Promega), as recommended by the supplier. Control reactions were also prepared to check for possible exogenous contaminants. The samples were incubated in an Arktik thermocycler (Thermo Fisher Scientific) and the conditions used were 25 °C for 5 min, 42 °C for 60 min and 70 °C for 15 min. cDNA was stored at -20 °C for further amplification. The transcripts of BCL2 apoptosis regulator (BCL-2), Caspase 3 (CASP3), Autophagy-related 5 (ATG5) and Autophagy-related 7 (ATG7) genes were quantified by qPCR, using the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as reference (Table 2). For each target, a relative standard curve was constructed to validate the comparative Cq method. Reactions were prepared to a final volume of 10 μL containing cDNA, primers and GoTaq qPCR Master Mix (A6001, Promega), and carried out in the 7300 Real Time PCR System (Applied Biosystems) equipment. Cycling conditions were denaturation of 95 °C for 1 min followed by 40 cycles with denaturation at 95 °C for 15 s and annealing/amplification a 60 °C for 1 min.

Genes
Sequences 5′-3′ www.nature.com/scientificreports/ tin structure and staining. The percentages of early apoptosis, late apoptosis and necrosis were determined from the total number of counted cells as previously described 82,83 . Autophagy assay. In a sterile 24-well culture plate, the Sarcoma 180 tumor cells (2 × 10 5 cells/well) were treated with complex 1 in three concentrations (1 µM, 4 µM and 20 µM), hydrogen peroxide 1 mmol/L (positive control) or culture medium (negative control). The cells were treated for 24 h and afterwards Monodansylcadaverine (MDC)-Sigma-Brazil-(0.05 mM/well) was added and incubated at 37 °C, CO 2 5% for 60 min in the darkness. The samples were collected, centrifuged and the pellet washed three times with ice-cold PBS 1×. The pellet was resuspended in a solution of PBS + Glycine (0.1 M) and 500 µL of this solution of cells-PBS-Glycine was placed in a 24-well plate and immediately analyzed under a fluorescence microscope (Zeiss L5M510, Germany). Four microscopic fields were randomly analyzed at 200× magnification and fluorescence intensity was captured by Image J software.

BCL
Statistical analysis. The IC 50 or Half Maximal Inhibitory Concentration (concentration that inhibits 50% of cell growth) was determined using the program GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, California, USA), from a non-linear regression, where the percentage of cell viability was determined as a logarithmic function of tested concentrations, assuming a 95% confidence interval (p < 0.05). The statistical analyses of cell cycle progression and autophagy assays were performed with one-way ANOVA with Bonferroni's multiple comparisons post-test. Statistical analyses for ROS and apoptosis assays were performed with two-way ANOVA with Bonferroni's multiple comparisons post-test. The lipidic peroxidation analysis was performed by T-test. Data were considered statistically significant when p < 0.05. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.